Page 1 of 32

Working for a better future



### REPORT

: Acute Toxicity t o Rainbow Trout

HLS study number: CEF0010 Version ID: Audited Draft 1 Report Issue date: 10 October 2013Huntingdon Life Sciences: CEF0010

2 

## Sponsor and Te st Facility Details Sponsor

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences: CEF0010

3 

## Ta ble of Content

s Sponsor and Test Facility Details

................................ ................................ .......................... 2 Compliance with Good Laboratory Practice

................................ ................................ .......... 5 Quality Assurance Statement

................................ ................................ ................................ 6 Contributing Scientists

................................ ................................ ................................ .......... 7 Summary

................................ ................................ ................................ .............................. 8 1. Introduction

................................ ................................ ................................ ................... 9 2. Test Substance

................................ ................................ ................................ ........... 10 3. Experimental Procedure

................................ ................................ .............................. 11 3.1 Test species

................................ ................................ ................................ .......... 11 3.1.1 Name

................................ ................................ ................................ ............. 11 3.1.2 Source

................................ ................................ ................................ ........... 11 3.1.3 Acclimatisation

................................ ................................ ............................... 11 3.2 Diluent water

................................ ................................ ................................ ......... 11 3.3 Test substance preparation

................................ ................................ ................... 12 3.4 Exposure conditions

................................ ................................ .............................. 12 3.4.1 Experimental design

................................ ................................ ...................... 12 3.4.2 Test concentrations

................................ ................................ ........................ 12 3.4.3 Medium renewal

................................ ................................ ............................ 12 3.4.4 Stability of test concentrations

................................ ................................ ....... 12 3.4.5 Environmental conditions

................................ ................................ ............... 13 3.5 Criteria of effect

................................ ................................ ................................ ..... 13 3.6 Evaluation of data

................................ ................................ ................................ . 13 3.7 Protocol deviations

................................ ................................ ................................ 13 4. Maintenance of Records

................................ ................................ .............................. 14 5. Results

................................ ................................ ................................ ........................ 15 5.1 Chemical analysis

................................ ................................ ................................ . 15 5.2 Mortality and observations

................................ ................................ .................... 15 5.3 Environmental parameters

................................ ................................ .................... 15 6. Conclusions

................................ ................................ ................................ ................. 16 

## List of T ables Table 1 Measured concentrations

................................ ................................ ............. 17 Table 2 Sub
- lethal effects

................................ ................................ ......................... 18 Table 3 Environmental parameters

................................ ................................ ........... 19Huntingdon Life Sciences: CEF0010

4 

## List of A ppendices Appendix 1 Certificate of analysis

................................................................................... 20 Appendix 2 Typical water quality characteristics of the dil uent supply

............................. 21 Appendix 3 The determination of in dechlorinated tap water

.......................... 22 Appendix 4 Compliance statement

................................................................................. 32Huntingdon Life Sciences: CEF0010

5 

## Compliance with Good Laboratory Practice
: Acute Toxicity to Rainbow Trout The study described in this report was conducted in co mp liance with the fo llo wing Good Laboratory

Practi ce standards and I consider the data generated to be valid. The UK Good Laboratory Practi ce Regulat io ns (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994). OECD Principles of Good Laboratory Practi ce (as revised in 1997), ENV/MC/CHEM (98) 17. EC Commissio n Di

rective 2004/10/EC of 11 February 2004 (Official Journal No L 50/44). These princi

ples of Good Laboratory

Practice are accepted by

the Regulatory

A uthori ti es of th e United States of America and Japan on the basis of Intergovernmental Agreements. No claim of com pliance is made for the results of analysis of the diluent water supply.

________________________________ ___________ ________________________ Romalie Allen BSc Date Study Di rector Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0010

6 

## Quality Assurance Statement
: Acute Toxicity to Rainbow Trout The fo llo wing inspect io ns and audits have been carried out in relation to this study: Study Phase Date(s) of I nspection Date of Reporting to Study Director and Management Protocol Audit 13 Jun 2013 13 Jun 2013 Report Audi

t 10 Oct 2013 10 Oct 2013 Process based inspections : At o r about th e time this study w as in progress inspect io ns of procedures em

ploy ed on this ty

pe of study

w ere carri ed out. T hese were conducted and reported to appropriate Company Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Envi ronmental

m easurements & mo nit oring/Ammo n ia analysis 07 Mar 2013 07 Mar 2013 Study management & conduct 26 Jun 2013 26 Jun 2013 Sam pling of te st media for chemical analysis 09 Jul 2013 09 Jul 2013 Dose Form

ulation 09 Jul 2013 09 Jul 2013 Study management & conduct 09 Jul 2013 09 Jul 2013 Ex perimental

Set -up 11 Jul 2013 11 Jul 2013 Experimental Set -up 08 Aug 2013 09 Aug 2013 Sam pling of tes t media for chemical analysis 08 Aug 2013 09 Aug 2013 Dose Form

ulation 08 Aug 2013 16 Aug 2013 Study management & conduct 09 Aug 2013 16 Aug 2013 O bservations 13 Sep 2013 13 Sep 2013 Measurements 10 Oct 2013 10 Oct 2013 Standard preparation & Fortificat io n procedures 05 Aug 2013
- 06 Aug 2013 07 Aug 2013 Results Processing 30 Aug 2013 30 Aug 2013 Chro mat ography 30 Aug 2013 30 Aug 2013 Sam ple pr eparation (to i

nclude sample management & tracking) 30 Sep 2013 30 Sep 2013
In addit io n, an inspect io n of the facilit y where thi s study was conducted was carried out on an annual basis. These inspect io ns were reported to Company Management.

.......... .............................................................................. ..................... T Pullinger MRQA Date Group Manager Department of Qualit y Assurance Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0010

7 

## Contributing Scientist s
: Acute Toxicity to Ra inbow Trout 

### Study management Aquatic Ecotoxicology and Biodegradation Romalie Allen BSc Study Di rector 

### Expert department Environmental Analysis Simo n Tate Higher National Certificate (Applied Chemistry) Senior Study

ManagerHuntingdon Life Sciences: CEF0010

8 

## Summary A study was performed to assess the acute toxicit y of to rainbow trout ( Oncorhynchus mykiss ) under semi

- static condi ti ons wi th daily renewal o f th e media. The study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex to Commissio n Regul at io n (EC) No 440/2008 (O.J. No. L142/446, 2008) Part C, Method 1 “Acute Toxicit y For Fish” and the OECD Guideline for Testing of Chemicals No. 203 “Fish, Acute Toxicit y Test” (1992). Groups of seven juvenile fish were exposed to , dispersed in wate r at nominal concentrations of 1.17, 2.58, 5.68, 12.5 and 27.5 mg/L

. The test media were prepared by d ir ect addi

ti on of the test substance to water fo r the 27.5 m g/L preparation and by dilution of thi s high concentration (aliquots of 850 mL, 1880 mL, 4.13 L and 9.09L) as appropriate

. At all concentrations, the intended exposure levels of were achieved between 70 to 98 % of their no minal values . The slight ly lo w concentrati ons are considered to have resul ted f rom a slight ly lo wer sol

ubili ty at the lo wer temperature fo r an acute fish study when com pared wi th the physi ochemical properti es study (20oC wi th approx. 15oC for thi s fish study ). The concentrations were ma int ained during the test (between 79 to 136 % of their starting values). B ased on a ge ometric mean, overall mean measured levels were 0.95 0 , 2.19, 4.65, 10. 5 and 24. 5 mg/L and these values have been used in the determination of study end

points. Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of exposure . No mortalit y occurred during the 96 hour test, therefore the 96 hour LC 50 was greater than 24.5 mg/L under the condi ti ons of this study

. The ‘no

- observed effect concentration’ (NOEC) was 10.6 mg/L as t reatment - related effects (loss of body

colour, loss o f co - ordinat io n and /or sp ir al swimming) were exhibited at measured concentrations 24.5 mg/L at 24 hours.Huntingdon Life Sciences: CEF0010

9 

## 1. Introduction This study

was designed to assess the acute toxicit y of to rainbow trout ( Oncorhynchus mykiss ) under semi

- static condi ti ons. Th e study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex to Commissio n Regulat io n (EC) No 440/2008 (O.J. No. L142/446, 2008) Part C, Method 1 “Acute Toxicit y For Fish” and the OECD Guideline for Testing of Chemicals No. 2 03 “Fish, Acute Toxicit y Test” (1992). The protocol was approved by Huntingdon Life Sciences Management and the Study Di rector on 17 June 2013 and by the Sponsor on 1 October 2013. The study was conducted at Huntingdon Life Sciences, Eye Research Centre, E ye, Suffo lk, IP23 7PX, England. The experimental start and complet io n dates of the study were 25 June 2013 and 30 August 2013 , respectively. A study to determine the physiochemical propert ie s of ( HLS study : CEF0001) indicated that the limit of so l ubili ty of the test substance was 27.5 mg/L at 20oC.Huntingdon Life Sciences: CEF0010

10 

## 2. Te st Substance
Name Chemical name CAS No. Physical state Powder Batch number 12342 Puri ty To be confirmed Storage Room tem perature in the dark Expi ry Date To be confirmed Date received: 4 March 2013 Certificate of analysis: Appendix 1Huntingdon Life Sciences: CEF0010

11 

## 3. Experimental Procedure 3.1 Test species 3.1.1 Name Rainbow trout ( Oncorhynchus mykiss ). 3.1.2 Source The fish were supplied by a commerc ial fish farm in the UK and were reared at the farm fro m eggs th at hatched in March 2013

. 3.1.3 Acclimatisation The stock of fish was obtained fro m th e supplier on 4 July 2013 and they were held in an aerated supply of diluent water under flow

- th rough condi

ti ons unt il use. During the 14

- day peri od immediately before the definit ive test, temperatures remained within the range 14.2 to 14.5  C, pH values wit hin the range 7.51 to 7.60 , disso lved oxygen concentrations within the range 65 to 95 % ai

r saturati on val

ue (AS V) and total hardness within the range 143 to 227 mg/ L as CaCO 3 (see deviations f rom Protocol sect io n)

. The fish were fed daily

wi th an amount of

commerci al fish food (TROUW (UK) Ltd; Nutra Fr y 02) equivalent to between 1 and 4% of the total wet

- weight o f fish in the ho ld ing tank. No f ood was given during the 24

- hour peri od immedi ately before exposure or during the exposure period itself. No medication was given during the holding period, and mortalit y was recorded as <0.5 % in the 14 days before the def init ive test. The size of the fish used in the definit ive test was determined by weighing and measuring a sample of ten fish taken at random fro m th e ho ld ing tank on 15 August 2013 ; their mean total length was 6.5 cm

and their mean wet weight was 2.52 g. 

### 3.2 D iluent water The water used to hold the fish and for the study was laboratory

tap water, dechlorinated and softened by

passage through a water purificat io n system. It was passed through a high grade activated carbon filter to remove chlorine and any organ ic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply . The two grades of dechlorinated water were then remixed to give a supply

wi th the desi red w ater hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gent ly aerated before being supplied to the ho ld ing and test areas. Ty pical water qualit y characteri st ic s of the diluent supply are give n in Appendix 2
.Huntingdon Life Sciences: CEF0010

12 

### 3.3 Test substance preparation The method of preparation used during the definit ive test was based on the results of a fo rm ulat io n tri al and a range finding test. The test substance ( 1.1 g) was ground into a smo oth pourable paste on an agate mortar with th e dropwise addit io n of dechlorinated tap water . The paste was then added to a 40 L tank containing dechlorinated tap water , made up to volume and sti rred f or a minimum of one hour. The formulation was then fil tered (0.45 μm

pore size) to provide an aqueous sol ut io n wi th a no minal concentration of 27. 5 mg/L that was ei

th er used di

rect ly fo r the highest test concentration (20L) or a liquots ( 850 m L, 1880 m

L, 4.13 L or 9.09L ) were diluted in tanks (20L capacit y) us ing dechlorinated tap water to provide test media with nominal concentrations of

12.5, 5.68, 2.58 and 1.17 mg/L. 

### 3.4 Exposure conditions 3.4.1 Experimental design A range finding test was fo llo wed by a definit ive test wi th five test concentrations, pl us a diluent water control group

. In the definit ive test, seven fish were placed at random into each glass aquarium containing th e prepared control or test media. Each vessel contained 20 lit res of medium to a depth of 19 cm . This provided an init ial static loading of 0.882 g bodyweight/Litre. 3.4.2 Test concentrations The range finding test emplo yed no minal test concentrations of 0.27, 2.7 and 27 mg/ L. After 96 hours, one fish died at 0.27 m g/L, which was considered not to be treatment related since no mortali

ti es were observed at th e two higher test concentrations. At 27 mg/L, there were no treatment - related effects noted by 72 hours, but at 96 hours , all fish showed let hargy . No significant treatment - related effects were noted in the other test groups. Bas ed on thes e resul ts, the definit ive test emplo yed the f ol lo wing no minal concentrations of 1.17, 2.58, 5.68, 12.5 and 27.5 mg/L ; the highest concentration emplo yed approximated to the limit of aqueous sol

ubilit y of the test substance

. 3.4.3 Medium renewal The fish were exp osed to the control or test condit ions for a period of 96 hours with daily batch renewal o f th e media to ensure the maintenance o f satisfactory environmental condi ti ons and to maintain stable exposure levels. 3.4.4 Stability of test concentrations The test conce nt rations of were measured using liquid chromatography wi th tandem m ass spectrometric detection (LC

- MS/MS, see Appendix 3 ). Two , mid

- vessel samples ( 10 mL) of media were taken fro m th e control and test vessels at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis. On each occasio n, one of the samples was analysed and the other was stored in a freezer in case further analysis was required.Huntingdon Life Sciences: CEF0010

13 3.4.5 E nvironmental conditions Treatment and control g roups were maintained at 12 to 17  C throughout the exposure period and constant to within  1  C during the study . The tem perature of the water in the diluent water control vessel was continuously mo nit ored during the study

. Supplementary

aeration was prov id ed via narrow bore glass tubes. A photoperiod of 16 hours light: 8 hours dark was maintained, with periods of subdued light ing at the beginning and end of each light phase. Daily records of temperature, pH and disso lved oxygen were kept for each contro l and test vessel together with measurements of total hardness for selected vessels at 0 hours. The fish were not fed during the 96 hour exposure period. 

### 3.5 Criteria of effect The cri teri a of death em ploy ed in this study

were (i) absence of respiratory mov em ent and (ii) absence of response to physical st imulat io n of the caudal peduncle. In addit io n to observat io ns on mortalit y at approximately 2, 4, 24, 48, 72 and 96 hours, subjective assessments were also made on the incidence and t ype of any sub

- let hal effe cts com pared wi th control f ish. 

### 3.6 Evaluation of data The “no observed effect concentration” (NOEC) was derived by direct inspect io n of the data fo r lethal and treatment - related

- effects. An incidence rate of more than one affected fish out of seven is consid ered to be significant. 

### 3.7 Protocol deviations The fo llo wing deviat io n fro m Protocol occurred: The final study

Protocol states that “Fish to be used in the test will be held for at least 7 days in water of the qualit y and temperature to be used in the test”. H ardness measurements should be approximately 120 to 180 m

g/L as CaCO 3 . On Day
- 6 the hardness measurement was 227 mg/L as CaCO 3 fo r the batch of fish available for use on this study . All

other recordings for this parameter were within specificat io ns and on Da y 0 was 164 mg/L as CaCO 3 . Therefore the fish were considered suitable for use on this study

. This deviation fro m protocol i s considered not to affect the integrity or validi ty of the study

.Huntingdon Life Sciences: CEF0010

14 

## 4. Ma intenance of Records This report has been com piled from

original data, which will be stored, together with a copy of the final report, a copy

of the protocol

and amendments in Hunt ingdon Life Sciences’ Archives, Huntingdon Life Sciences Ltd, Eye, Suffol k, UK. Records will be retained for a minimum pe ri od of at least one year from the date of issue of th e final report. At the end of the one year retention peri od the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any i tem be di scarded wi thout the Sp onsor’s knowledge. Hunt ingdon Life Sciences will retain the Qualit y Assurance records relevant to this study f or twenty y ears, and a copy

of the final report in its archives indefinitely

.Huntingdon Life Sciences: CEF0010

15 

## 5. Results 5.1 Chemical a nal ysis The results of chemical analysis are given in Table 1 and example chromatograms are given in Appendix 3
. At all concentrations, the intended exposure levels of were achieved between 70 to 98 % of their no minal values . The sl ight ly lo w concentrati ons are considered to have resul ted f rom a slight ly lo wer sol

ubili ty at the lower tem

perature for an acute fish study when com pared wi th the physi ochemical properti es study (20oC wi th approx. 15oC for thi s fish study ). The concentrat io ns were ma int ained during the test (between 79 to 136 % of their starting values). Based on a geometric mean, overall mean measured levels were 0.950, 2.19, 4.65, 10.5 and 24.5 mg/L and these values have been used in the determination of study end

points

. 

### 5.2 Mortality and observations No mortalit y occurred during the 96 hour test, therefore the 96 hour LC 50 could not be calculated but m

ust be greater than 24. 5 mg/L. A chronological record of treatment

- rel ated effects is given in T able 2
. One fish was af fected at 0.95 0 , 4.65 , 10. 5 and 24. 5 mg/L during most of the test ; addit io nally at 24. 5 mg/ L, th ree fish were affected at 24 hour s only. Treatment

- related effects comprised hypervent ilat io n, lo ss of body colour, loss of co

- ordi

nat io n and spiral swimming. The ‘no

- observed effect concentration s ’ (NOEC) was 10.5 mg/ L and the ‘lowest observed affect concentration’ (LOEC) was 24.5 mg/L. 

### 5.3 Environmental parameters The measurements of water qualit y (tem perature, pH, concentrations of dissolved oxygen and total hardness) are summarised in Table 3 ; they remained wi

th in acceptable limits throughout th e study wi

th the except io n of total hardness befo re commencement of the test (see deviat io ns fro m Protocol

sect io n)

. At all concentrations, the test media were colourless.Huntingdon Life Sciences: CEF0010

16 

## 6. Conclusions T he acute toxicit y of

to rainbow trout ( Oncorhynchus mykiss ) has been assessed, under semi

- static condi

ti ons. No mortalit y occurred during the 96 hour test; th erefore , th e 96 hou r LC 50 could not be calculated but was considered under the condit io ns of this study to be greater than 24. 5 mg/L. The 96 hour ‘no

- observed effect concentration’ (NOEC) is considered to be 10.6 mg/L.Huntingdon Life Sciences: CEF0010

17 Table 1 Measured concentrat ions

Nominal conc entrations (mg/ L) Measured concentrations Overall mean* (mg/L) 0 hours (F) 24 hours (E) % t 0 72 hours (F) 96 hours (E) % t 72 mg/ L %N mg/ L %N mg/ L %N mg/ L %N Control nd

- nd

- - nd

- nd

- - nd 1.17 0.9 56 82 0.962 82 101 0.850 73 1.04 89 122 0.950 2.58 2.16 84 2.16 84 100 2.11 82 2.33 90 110 2.19 5.68 4.93 87 4.4 4 78 90 4.42 78 4.82 85 109 4.65 12.5 12.1 97 9.6 1 77 79 8.77 70 11.9 95 136 10.5 27.5 27.0 98 23.7 86 88 21.2 77 26.6 97 125 24.5
nd None detected (less than the limit of detection = 0. 04 mg/L ; Appendix 3 ) % ti Measured concentration after 24 or 96 hours expressed as a percentage of the starting concentration (0 and 72 hours) F Freshly prepared medium E Expired medium %N Measured concentration expressed as a percentage of the nominal concentration (cal culated using unrounded value) * Geometric mean

- No measurement requiredHuntingdon Life Sciences: CEF0010

18 Table 2 Sub - lethal effects
concentrations (m g/L) Abnormality Initial population = 7 fish / concentration
N ominal M easured 2 h 4 h 24 h 48 h 72 h 96 h Control nd No abnormalities detected 7/7 7/7 7/7 7/7 7/7 7 /7

1.17 0.950 No abnormalities detected 6/7 5/7 6/7 6/7 6/7 6/7

Hyperventilation 0/7 1/7 0/7 0/7 0/7 0/7
2.58 2.19 No abnormalities detected 7/7 7/7 7/7 7/7 7/7 7 /7

5.68 4.65 No abnormalities detected 7/7 6/7 6/7 6/7 6/7 6/7

Darkened pigmentation 0/7 1/7 1/7 1/7 1/7 1/7

12.5 10.5 No abnormalities detected 7/7 5/7 6/7 5/7 5/7 5/7

Darkened pigmentation 0/7 1/7 0/7 0/7 0/7 0/7 Curved spine 0/7 1/7 1/7 1/7 1/7 1/7

27.5 24.5 No abnormalities detected 6/7 4/7 3/7 4/7 4/7 4/7

Darkened pigmentation 0/7 1/7 1/7 0/7 0/7 0/7 Loss of body colour 0/7 1/7 1/7 1/7 1/7 1/7 Loss of co

- or dination 0/7 0/7 3/7 0/7 0/7 0/7 Spiral swimming 0/7 0/7 3/7 0/7 0/7 0/7 x/y Number of fish affe cted / number of fish surviving nd None detected (less than the limit of detection = 0. 04 mg/L ; Appendix 3 )Huntingdon Life Sciences: CEF0010

19 Table 3 Environmental parameters

concentrations (mg/L) Temperature o C pH Dissolved oxygen % ASV Total

hardness (mg/L)* N ominal M easured Min Max Min Max Min Max 0 hours Control nd 14.5 15.7 7.65 8.05 93 101 161 1.17 0.952 14.1 15.7 7.7 2 8.17 94 102 - 2.58 2.19 14.1 15.7 7.7 9 8.25 95 101 - 5.68 4.65 14.2 15.7 7.78 8.26 94 101 - 12.5 10.6 14.5 15.7 7.80 8 .27 94 100 - 27.5 24.6 14.8 15.8 8.01 8.26 94 101 163
ASV Air saturation value (100% ASV = 9.09 mgO 2/L at 20oC). * As CaCO 3 nd None detected (less than the limit of detection = 0.04 mg/L; Appendix 3 ) Continuous monitoring of vessel media temperature = 14. 1 to 1 6.0 °CHuntingdon Life Sciences: CEF0010

20 Appendix 1 Certificate of analysis Aw aiting supply from sponsorHuntingdon Life Sciences: CEF0010

21 Appendix 2 Ty pical water quality characteristics of the diluent supply Diluent water com

prised filtered dechlorinated tap water blended with tap water that had been so ft ened and subsequently treated by reverse osmosis to a hardness of 150

- 200 m g/L as CaCO 3 . Analysis of a sample taken in January 2013 gave the following results:
Colon y count 37°C 0 / 1 mL Coliform bacteria / 100mL 2 / 100 mL E. coli 0 / 100 mL μg/L Organochlorine pesticides <1.24 Organophosphorus pesticides <3.30 Pol ychlorinated biphenyls <0.007 mg/L Aluminium <0.0200 Ammonium (as NH 4 ) <0.01 Arsenic <0.0020 Boron 0.0296 Cadmium 0.00031 Calcium 51 Carbon (total organic) 0.55 Chloride 18.7 Residual disinfectant (total chlorine) <0.02 Residual disinfectant (free chlorine) <0.02 Chromium <0.0020 C.O.D. 3.62 Cobalt <0.0020 Copper 0.0248 Fluoride <0.05 Iro n <0.015 Lead 0.0006 Magnesium 3.010 Mercury 0.00008 Nitrate (as NO 3 ) 4.66 Nitrite (as NO 2 ) <0.05 Nickel 0.00309 Phospho rus <0.10 Potassium 1.650 Silver 0.00180 Sodium 8.360 Tin <0.002 Suspended solids <2.0 Zinc 0.0198 Conductivity (20°C) 341 μS/cm Turbidity <2.0 NTU
NTU Nephelometric turbidity units.
No claim of com pliance is made for the above data.Huntingdon Life Sciences: CEF0010

22 Appendix 3 The determination of in dechlorinated tap water 

## 1. Method 1.1 G eneral principle Test sol ut io ns were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) pri or to quant it ation by liquid chromatography wi th tandem m ass spectrometric detection system (LC

- MS/MS). Materials Grade Actonitrile HPLC Ammonium form ate AR Formic acid AR Methanol HPLC Water HPLC 1.1.1 Test article fortifying and calibration solutions Some standard sol

uti ons used in this study

have been shared wit h other GLP studies being performed f

or the same sponsor. This is considered to have no impact on the integrit y or validit y of

this study

. A weighed amount of was di ssolved in acetonitrile to produce a 1 mg/mL stock standard solut io n. The stock standard was diluted progressively wit h met hano l to give fo rtificat io n standard soluti ons. The appropri ate fortificat io n standard so lut io n was further diluted with water:acetoni trile:formic acid (95:5:0.1 v:v:v) to produce a series of calibrat io n sol ut io ns in the range 0.1 to 5 ng/mL. 1.1.2 Ty pical preparation of reagents Preparati on of water:acetonitrile:formic acid (95:5:0.1 v:v:v) : Acetonitrile (5 0 mL) and formic acid (1 mL) w ere thoroughly mixed with water ( 95 0 mL) pri or to use. Preparati on of

water:methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammo nium formate: Water (900 mL) was thoroughly mixed wi

th methano l (100 mL), formic acid (1 mL) and ammo nium formate (0.63 g) prior to use. Preparati on of

0.1% formic acid in methano l: Methano l (1000 mL) was thoroughly mixed with formic acid (1 mL) prior to use. NOTE

– to prepare different vol umes of the above reagents, the ratio of the individual com ponents was maintained.Huntingdon Life Sciences: CEF0010

23 

### 1.2 Procedure An aliquot (10 mL) of the sample was taken and fortified if required. Water:acetonitrile:formic acid (95:5:0.1 v:v:v) (10 mL) was added and mixed well . The sample was diluted by

a factor of 200 wi

th water:acetonitrile:formic acid (95:5:0.1 v:v:v)

. Any further dilut io ns, where required, were performed with water:acetoni tri le:formic acid (95:5:0.1 v:v:v) pri or to quanti tation by L C
- MS/MS. 1.2.1 Instrumentation LC - MS/MS conditions Instrument: AB Sciex API 4000 Data system: Analyst v.1.4.2 Ioni sat io n mo de: Ionspray posi tive Ion moni toring details: MRM m/z 186>57 Col umn: Pheno me nex aqua C 18 (5 cm  2 mm) Mobile phase A: Water:methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammo nium formate Mobile phase B: Methano l + 0.1% formic acid (by v ol ume) Pum p programme: 60:40 A:B isocratic Cycle time: 8 minutes Flow rate: 0.3 m L/min Inject io n vo lu me: 20 μL Retention t ime: Approximately 1. 4 minutes LOQ: 0. 1 m g/L LOD: 0.1 ng/m L (equivalent to 0.04 mg/L in aqueous media)Huntingdon Life Sciences: CEF0010

24 1.2.2 Calculation of results Test samples were quant ified using the fo llo wing equati on: Residue found (mg/L) = 1000 1 x D x M 1 x x Where x (residue concentration in final so lut io n) was calculated using the l inear regression:
y = m x + c where x (concentration in ng/mL) = y  c m c = int ercept m = slope y = peak area of sample M = matri

x concentration (mL/mL) D = dilut io n factorHuntingdon Life Sciences: CEF0010

25 

## 2.0 Results and Discussion 2.1 Calibration The chro mat ographi c re sponse to was shown to be linear over the range of concentrations 0.1 to 5 ng/mL. Ty pical calibrat io n data is presented in Table 1 and Figure 1. Ty pical chromatograms of calibrat io n standards are presented in Figures 2 and 3. 

### 2.2 Validation The methodology

f or the determinat io n of residues in dechlorinated tap water was validated at 0.1 m

g/L and 3 0 m

g/L. The validat io n data are presented in Table 2 and summarised in the fo llo wing table:

Fortification level (mg/L) Mean recovery (%) CV (%) 0.1 96 1.4 3 0 94 4.9 ( 95 ) ( 3.5 ) ( ) O verall values 

### 2.3 Limit of quantitation (LOQ) The limit of quant it ation is defined as the lowest fortificat io n level at which acceptable recovery data are obtained. The validation of the methodology fo r the determination of residues of in dechlorinated tap water , demonstrated that i t can be accurately determined at a limit of quant it ation (LOQ) of 0.1 mg/L. 

### 2.4 Limit of detection (LOD) The limit of detection of the method is defined as the val ue of the lowest calibrat io n standard chro mat ographed that gave a peak height to baseline no ise ratio  3. For this study the limit of detection was 0.1 ng/m

L (equivalent to 0. 04 m

g/L in dechlorinated tap water ). 

### 2.5 Samples Sam ples o f dechlorinated tap wa ter from the test were analysed in suitably sized batches along wi th control samples fortified wit h which acted as procedural recovery samples. Procedural recoveries performed on control dechlorinated tap water were in the range 70 to 110% confirm ing the validit y of the methodology

on each day of analysis. Ty pical chromatograms of untreated, untreated fortified with and treated sam ples are presented in Figures 4 to 7.Huntingdon Life Sciences: CEF0010

26 Table 1 Ty pical standard calibration for by LC - MS/MS

Standard co ncentration (ng/m L ) Peak area

0 0 0.1 1.09054e+004 0.2 1.65429e+004 0.4 3.34644e+004 0.8 6.64350e+004 1 7.38713e+004 2 1.51181e+005 3 2.46303e+005 4 3.13915e+005 5 3.93727e+005 Calibration curve: y

= 78773.8 x + 657.708 (batch 4 ) r = 0.9 9 9 5 Where x = concentration y = peak area r = regression coefficientHuntingdon Life Sciences: CEF0010

27 Table 2 Validation of in dechlorinated tap water by LC - MS/MS

Fortification level Replicate Analyte Detected Recovery Mean Standard Deviation Relative

Standard Deviation Uni t: mg/L Unit: mg/L % % % % Control A ND
- Control B ND
- 0.1 A 0.0972 97 0.1 B 0.0941 94 96 1.3 1.4 0.1 C 0.0958 96 0.1 D 0.0945 95 0.1 E 0.0973 97 30 A 29.1 97 30 B 28.6 95 94 4.6 4.9 30 C 26.3 88 30 D 27.5 92 30 E 29.9 100 Overall mean (%) 95 Overall standard deviation (%) 3.3 Overall relative standard deviation (%) 3.5 General Notes: ND

– Not detected (<LOD of 0.04 mg/L) Control aquatic media sample used

– 13/0 0/A3234 Data system: Analyst v.1.4.2Huntingdon Life Sciences: CEF0010

28 Figure 1 Ty pical standard chemical calibration for by

LC - MS/MS

CEF0010_B4.rdb : "Linear" Regression ("No" weighting): y = 78773.8 x + 657.708 (r = 0.9994752)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Concentration, ng/mL 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 4.0e5Area, countsHuntingdon Life Sciences: CEF0010

29 Figure 2 Ty pical chromatogram of a 0.1 ng/mL calibration

standard

Sample Name: "Cal 0.1 ng/mL" Sample ID: "" File: "CEF0010_B414.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 0.100 ng/mL Calculated Conc: 0.130 ng/mL Acq. Date: 19/08/2013 Acq. Time: 17:43:14 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.41 min Use Relative RT: No Int. Type: Valley Retention Time: 1.43 min Area: 1.09054e+004 counts Height: 1.68e+003 cps Start Time: 1.33 min End Time: 1.63 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps

1.43 1.48 5.24 1.37 4.55

2.04 3.61 5.43 4.81 6.92 7.45 5.04 1.66 7.60 5.87 2.83 7.12 3.86 5.68 2.27 6.54 4.44 6.87 3.07 1.16 5.99 4.00 3.21 2.41 6.47 0.91 0.14 0.38 7.77

0.70

Figure 3 Ty pical chromatogram of a 5 ng/mL cali bration

standard

Sample Name: "Cal 5 ng/mL" Sample ID: "" File: "CEF0010_B420.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 5.00 ng/mL Calculated Conc: 4.99 ng/mL Acq. Date: 19/08/2013 Acq. Time: 18:35:16 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.41 min Use Relative RT: No Int. Type: Valley Retention Time: 1.44 min Area: 3.93727e+005 counts Height: 5.82e+004 cps Start Time: 1.26 min End Time: 1.85 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps 1.44

1.66 7.89 1.25 7.67 5.69 5.88 2.80 5.00 2.14 6.80

2.57 5.62 4.66 0.40 0.83 7.12 2.88 3.91 6.21 4.43 4.34 0.13 5.29 6.61 3.31 0.75Huntingdon Life Sciences: CEF0010

30 Figure 4 Chromatogram of an untreated dechlorinated tap water sample

(analytical identification 13/00/A3 2 81 Con)

Sample Name: "A3281 Con" Sample ID: "" File: "CEF0010_B419.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.00 ng/mL Acq. Date: 19/08/2013 Acq. Time: 18:26:35 Modified: No

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps

1.44

0.38 5.66 3.88 2.17 1.38 2.77 4.06 2.41 0.99 4.57 0.31 0.83 1.51 5.27 5.60 5.87 6.88 3.04 5.91 6.41 6.31 3.79 6.52 7.42 7.74 4.91 3.43 3.27 1.77 7.80 4.95 4.44 0.43 7.11 4.61 2.57

Figure 5 Chromatogram of an untreated dechlorinated tap water sample

fortified with at 10 m g/L (analytical ide ntification 13/00/A3281 F 10 )

Sample Name: "A3281 F10" Sample ID: "" File: "CEF0010_B421.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 26.8 ng/mL Acq. Date: 19/08/2013 Acq. Time: 18:43:57 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.41 min Use Relative RT: No Int. Type: Valley Retention Time: 1.44 min Area: 2.11570e+005 counts Height: 3.08e+004 cps Start Time: 1.27 min End Time: 1.78 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps 1.44Huntingdon Life Sciences: CEF0010

31 Figure 6 Chromatogram of a test sample at 12.5 m g/L of on

Day 0 (analytical identification 13/ CEF /A3 295 )

Sample Name: "A3295" Sample ID: "" File: "CEF0010_B425.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 30.1 ng/mL Acq. Date: 19/08/2013 Acq. Time: 19:18:36 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.41 min Use Relative RT: No Int. Type: Manual Retention Time: 1.44 min Area: 2.37925e+005 counts Height: 3.31e+004 cps Start Time: 1.26 min End Time: 1.92 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps 1.44

Figure 7 Chromatogram of a test sample at 12.5 m g/L of on

Day 1 (analytical identification 13/ CEF /A3352 )

Sample Name: "A3352" Sample ID: "" File: "CEF0010_B524.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 24.0 ng/mL Acq. Date: 20/08/2013 Acq. Time: 18:57:57 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.41 min Use Relative RT: No Int. Type: Valley Retention Time: 1.44 min Area: 1.72790e+005 counts Height: 2.69e+004 cps Start Time: 1.29 min End Time: 1.83 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4Intensity, cps 1.44

1.64 1.88 1.32 4.17 2.32 7.79 3.48 2.91 2.44 5.12 5.25

3.04 7.55 5.92 4.84 3.67 7.48

4.43 0.05 6.59 3.79 6.36 6.99 6.67 6.12 0.43 4.54

1.06 0.75Huntingdon Life Sciences: CEF0010

32 Appendix 4 C ompliance statement